Earl Avramis

Effects of MAPK and PI3K Pathways on PD-L1 Expression in Melanoma

Purpose: PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by melanoma driver mutations and modified by oncogenic signaling inhibition. Experimental Design: Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated. Results: No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1–exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines. Conclusions: In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1–exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies. Clin Cancer Res; 20(13); 3446–57. ©2014 AACR.

Adoptive Transfer of MART-1 T-Cell Receptor Transgenic Lymphocytes and Dendritic Cell Vaccination in Patients with Metastatic Melanoma

Purpose: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy. Experimental Design: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation. Results: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1–specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1–specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells. Conclusion: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. Clin Cancer Res; 20(9); 2457–65. ©2014 AACR.

Multifunctional T-cell Analyses to Study Response and Progression in Adoptive Cell Transfer Immunotherapy

UNLABELLED Adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) is a promising cancer treatment. Here, we investigate the in vivo functional activity and dynamics of the transferred cells by analyzing samples from 3 representative patients with melanoma enrolled in a clinical trial of ACT with TCR transgenic T cells targeted against the melanosomal antigen MART-1. The analyses included evaluating 19 secreted proteins from individual cells from phenotypically defined T-cell subpopulations, as well as the enumeration of T cells with TCR antigen specificity for 36 melanoma antigens. These analyses revealed the coordinated functional dynamics of the adoptively transferred, as well as endogenous, T cells, and the importance of highly functional T cells in dominating the antitumor immune response. This study highlights the need to develop approaches to maintaining antitumor T-cell functionality with the aim of increasing the long-term efficacy of TCR-engineered ACT immunotherapy. SIGNIFICANCE A longitudinal functional study of adoptively transferred TCR–engineered lymphocytes yielded revealing snapshots for understanding the changes of antitumor responses over time in ACT immunotherapy of patients with advanced melanoma.

Combination of pan-RAF and MEK inhibitors in NRAS mutant melanoma

BackgroundApproximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway.MethodsFourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated.ResultsThe majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3β and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines.ConclusionsThe combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.

COX-2 inhibition prevents the appearance of cutaneous squamous cell carcinomas accelerated by BRAF inhibitors.

Keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cuSCCs) develop in 15–30% of patients with BRAFV600E metastatic melanoma treated with BRAF inhibitors (BRAFi). These lesions resemble mouse skin tumors induced by the two‐stage DMBA/TPA skin carcinogenesis protocol; in this protocol BRAFi accelerates tumor induction. Since prior studies demonstrated cyclooxygenase 2 (COX‐2) is necessary for DMBA/TPA tumor induction, we hypothesized that COX‐2 inhibition might prevent BRAFi‐accelerated skin tumors. Celecoxib, a COX‐2 inhibitor, significantly delayed tumor acceleration by the BRAFi inhibitor PLX7420 and decreased tumor number by 90%. Tumor gene expression profiling demonstrated that celecoxib partially reversed the PLX4720‐induced gene signature. In PDV cuSCC cells, vemurafenib (a clinically approved BRAFi) increased ERK phosphorylation and soft agar colony formation; both responses were greatly decreased by celecoxib. In clinical trials trametinib, a MEK inhibitor (MEKi) increases BRAFi therapy efficacy in BRAFV600E melanomas and reduces BRAFi‐induced KA and cuSCC frequency. Trametinib also reduced vemurafenib‐induced PDV soft agar colonies, but less efficiently than celecoxib. The trametinb/celecoxib combination was more effective than either inhibitor alone. In conclusion, celecoxib suppressed both BRAFi‐accelerated skin tumors and soft‐agar colonies, warranting its testing as a chemopreventive agent for non‐melanoma skin lesions in patients treated with BRAFi alone or in combination with MEKi.

Natural Killer T Cells in Advanced Melanoma Patients Treated with Tremelimumab

A significant barrier to effective immune clearance of cancer is loss of antitumor cytotoxic T cell activity. Antibodies to block pro-apoptotic/downmodulatory signals to T cells are currently being tested. Because invariant natural killer T cells (iNKT) can regulate the balance of Th1/Th2 cellular immune responses, we characterized the frequencies of circulating iNKT cell subsets in 21 patients with melanoma who received the anti-CTLA4 monoclonal antibody tremelimumab alone and 8 patients who received the antibody in combination with MART-126–35 peptide-pulsed dendritic cells (MART-1/DC). Blood T cell phenotypes and functionality were characterized by flow cytometry before and after treatment. iNKT cells exhibited the central memory phenotype and showed polyfunctional cytokine production. In the combination treatment group, high frequencies of pro-inflammatory Th1 iNKT CD8+ cells correlated with positive clinical responses. These results indicate that iNKT cells play a critical role in regulating effective antitumor T cell activity.

Exposure to a Histone Deacetylase Inhibitor Has Detrimental Effects on Human Lymphocyte Viability and Function

Wong and colleagues show that human lymphocytes are highly susceptible to panobinostat, which alters their signaling pathways and is cytotoxic at a much lower dose than needed for antitumor activity. They caution against the combined use of panobinostat with immunotherapy for melanoma. Histone deacetylase inhibitors (HDACi) have been reported to increase tumor antigen expression, and have been successfully tested as adjuvants for melanoma immunotherapy in mouse models. In this work, we tested the effects of a pan-HDACi on human lymphocytes and melanoma cell lines. Effects of the pan-HDACi panobinostat (LBH589) on cell viability, cell cycle, apoptosis, and DNA damage were determined in peripheral blood mononuclear cells (PBMC) from 2 healthy donors, 13 patients with metastatic melanoma, 2 bone marrow samples from patients with different malignances, and 12 human melanoma cell lines. Intracellular signaling in lymphocytes, with or without cytokine stimulation, was analyzed by phospho-flow cytometry in one of each type. The IC50 in PBMCs was <20 nmol/L compared with >600 nmol/L in melanoma cell lines; >40% apoptotic cell death in PBMCs versus <10% in melanoma cell lines was seen at the same concentration. Phospho-histone variant H2A.X (pH2A.X) increased 2-fold in healthy donor PBMCs at 1 nmol/L, whereas the same effect in the melanoma cell line M229 required 10 nmol/L. pH2A.X was inhibited slightly in the PBMCs of 3 patients with metastatic melanoma at 1 nmol/L and in the melanoma cell line M370 at 10 nmol/L. Panobinostat inhibited phospho-STAT1/3/5/6, -p38, -ERK, -p53, -cyclin D3, and -histone H3 in flow cytometry–gated healthy donor B and T cells, whereas it induced up to 6-fold activation in patients with metastatic melanoma and bone marrow samples. In human lymphocytes, panobinostat alters key lymphocyte activation signaling pathways and is cytotoxic at concentrations much lower than those required for melanoma antitumor activity, resulting in an adverse therapeutic window. Cancer Immunol Res; 2(5); 459–68. ©2014 AACR.

PET imaging to non-invasively study immune activation leading to antitumor responses with a 4-1BB agonistic antibody

BackgroundMolecular imaging with positron emission tomography (PET) may allow the non-invasive study of the pharmacodynamic effects of agonistic monoclonal antibodies (mAb) to 4-1BB (CD137). 4-1BB is a member of the tumor necrosis factor family expressed on activated T cells and other immune cells, and activating 4-1BB antibodies are being tested for the treatment of patients with advanced cancers.MethodsWe studied the antitumor activity of 4-1BB mAb therapy using [18 F]-labeled fluoro-2-deoxy-2-D-glucose ([18 F]FDG) microPET scanning in a mouse model of colon cancer. Results of microPET imaging were correlated with morphological changes in tumors, draining lymph nodes as well as cell subset uptake of the metabolic PET tracer in vitro.ResultsThe administration of 4-1BB mAb to Balb/c mice induced reproducible CT26 tumor regressions and improved survival; complete tumor shrinkage was achieved in the majority of mice. There was markedly increased [18 F]FDG signal at the tumor site and draining lymph nodes. In a metabolic probe in vitro uptake assay, there was an 8-fold increase in uptake of [3H]DDG in leukocytes extracted from tumors and draining lymph nodes of mice treated with 4-1BB mAb compared to untreated mice, supporting the in vivo PET data.ConclusionIncreased uptake of [18 F]FDG by PET scans visualizes 4-1BB agonistic antibody-induced antitumor immune responses and can be used as a pharmacodynamic readout to guide the development of this class of antibodies in the clinic.

CRAF R391W is a melanoma driver oncogene

Approximately 75% of melanomas have known driver oncogenic mutations in BRAF, NRAS, GNA11 or GNAQ, while the mutations providing constitutive oncogenic signaling in the remaining melanomas are not known. We established a melanoma cell line from a tumor with none of the common driver mutations. This cell line demonstrated a signaling profile similar to BRAF-mutants, but lacked sensitivity to the BRAF inhibitor vemurafenib. RNA-seq mutation data implicated CRAF R391W as the alternative driver mutation of this melanoma. CRAF R391W was homozygous and over expressed. These melanoma cells were highly sensitive to CRAF, but not BRAF knockdown. In reconstitution experiments, CRAF R391W, but not CRAF WT, transformed NIH3T3 cells in soft-agar colony formation assays, increased kinase activity in vitro, induced MAP kinase signaling and conferred vemurafenib resistance. MAP kinase inducing activity was dependent on CRAF dimerization. Thus, CRAF is a bona fide alternative oncogene for BRAF/NRAS/GNAQ/GNA11 wild type melanomas.

Erratum to: Antitumor activity of the ERK inhibitor SCH722984 against BRAF mutant, NRAS mutant and wild-type melanoma

Deborah JL Wong and Lidia Robert contributed equally to this work. The online version of the original article can be found under doi:10.1186/1476-4598-13-194.